Niche Tet maintains germline stem cells independently of dioxygenase activity

Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal.

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- -----------------------------------------------Referee #1: In this study, the authors investigate the role of the Tet protein in Drosophila ovarian germline stem cell maintenance.They find that Tet is expressed in somatic cells and that RNAi knockdown specifically in the bab1+ population (which provide niche function for the GSCs) impairs GSC self-renewal.Further investigation reveals that this is due to a non-enzymatic scaffolding function of the tet protein that brings stat92E together with the BAP/PBAP complex protein Bap55.Building on a previous study that found that Jak/Stat signaling activates dpp expression in the bab1+ population, they show that RNAi knockdown of tet or the PBAP proteins (but not osa, which is specific for the BAP complex) reduces dpp expression, BMP signaling in GSCs, and GSC number.Rescue experiments using a wildtype or enzymatic dead allele of tet and in vitro co-IP experiments provide substantial support for the idea that tet has a non-enzymatic, scaffolding role in this process.Taken together, their data advance our understanding of the molecular mechanisms that contribute to Drosophila germline stem cell self renewal and the evidence for a non-enzymatic function of tet will be of interest more broadly.However, several concerns should be addressed before publication: 1.The data provided suggest that osa is required for GSC maintenance but not for dpp expression of BMP signaling within GSCs.What is the explanation for this?Also, since the conclusion from these data that the BAP complex is not required for dpp expression is based on this one RNAi line, the authors should confirm that the osa RNAi is, indeed, effectively knocking down osa transcript, as expected.2. The mass spectrometry approach associated with the data in Fig. 3A do not appear to be described in the methods.This should be added.3. Throughout the figures, it is not clear what is meant by "relative intensity".What are these intensity measurements relative to?The methods state that internal imaging controls were measured, but do not provide details about what these internal controls are.Also, the method of controlling for background signal (e.g. a background subtraction or correction) should be described.4. The method for unambiguously identifying CBs is not well described.Related to this, the object that the arrowhead in Fig. 1C is pointing to is not a very clear example of a rounded spectrosome that is clearly distinct from the other hts structures nearby.5.In Fig. S3, the Bap111 channel should be shown separately.The signal is dim and hard to appreciate in the multicolor image.In addition, these data show that tet is not essential for maintaining bap111 expression but do not provide strong support for the statement that Tet is not essential for maintaining the expression level of the whole BAP and PBAP complexes, as stated at the bottom of page 8.

Referee #2:
In this work, the authors report the function of the Tet protein in the Drosophila ovarian stem cell niche.They convincingly show that an enzymatic-dead Tet is necessary to ensure proper dpp transcription, an essential gene for germline stem cell (GSC) maintenance in the niche.Tet is required to bring together components of the PBAP complex and the Stat92E transcription factor.In their model, complexed Tet-PBAP-Stat bind to the dpp gene to activate its transcription and, thus, to regulate niche activity.I believe the biological question being investigated to be of interest.However, in my opinion the novelty of the findings appears somewhat limited as to warrant publication in a journal with a broad readership such as The EMBO J. Dioxygenase activityindependent Tet functions are well established in the field, as is the relationship between Jak/Stat and dpp transcription in the GSC niche.The MS does report some advancements, such as the demonstration of Tet-Stat92 and Tet-Bap55 physical interactions and the role of the complex in regulating dpp mRNA levels in the cap cells.Nevertheless, it fails to provide evidence for the binding of the complex to the dpp gene.Major points: 1-I have a serious issue with the current presentation of the MS.To follow is a list (by no means exhaustive) of some of the problems I detected: a) I have found typos and spelling mistakes in the text and figures (for instance, Fig 1L "reseuce" instead of "rescue"; Fig 2A enzyme, not ensyme).b) In several instances, the authors talk of "decrease GSCs" or "comparable CBs".I assume they refer to reduced GSC numbers or comparable CB numbers.c) Sentences with very similar contents are repeated throughout the text (for instance, in the Abstract: "Tet acts as a scaffold protein, bringing together the PBAP chromatin remodeling complex and the Stat92E transcription factor to activate the expression of BMP-like Dpp in the niche" and, two sentences later, "Mechanistically, Tet interacts directly with BAP55 and Stat92E, facilitating recruitment of the PBAP complex to the dpp promoter and activating Dpp expression".Or in the first paragraph of the Discussion "Tet functions as a protein scaffold to recruit the PBAP complex to Stat92E by directly interacting with Bap55 and Stat92E.Bap55-Stat92E can bypass the requirement of Tet in the niche to maintain BMP signaling and GSC self-renewal" and later in the paragraph "we propose that Tet acts as a protein scaffold, functioning independently of its enzymatic activity.It facilitates the interaction between the PBAP complex and Stat92E, leading to the activation of dpp expression in the niche".Or later in the Discussion "In this study, we show that TET functions as a protein scaffold independently of its enzymatic activity to recruit the PBAP complex to Stat92E to activate Dpp expression in the niche for controlling GSC selfrenewal in the Drosophila ovary.In this study, we have used a combination of genetics and biochemistry to demonstrate that Tet functions independently of its dioxygenase activity in the niche to control GSC self-renewal by regulating BMP signaling".d) There is one duplicated reference (Delatte et al 2016).e) Fig. S1 is missing from this version.f) The writing of the Fig. legends lacks detail and is redundant (every single panel containing statistical analyses has the following (or very similar) statement "Student's t test: ***p< 0.001; n. s., no significance".Or the repetition of the meaning of a dash line, arrows or arrowheads in all the panels of the same figure.This makes fig.legends very repetitive) g) Gene nomenclature is not consistent throughout (for instance, there is Bab1-Gal4, bab-Gal4, bab1-Gal4; or there is tet-RR-wt and tet-wt, tet-Dead and tet-ED) h) The full genotypes of the different experiments are not reported (what is "wt" in the different experiments?Canton S or Oregon flies?or Gal4 lines without UAS transgenes, or viceversa?).i) Treatments should also be clearly reported.The M+M section has a general statement that does not provide sufficient detail "newly eclosed flies at room temperature were cultured at 29{degree sign}C for the specified days before phenotypic analysis".As you may hint from my words, I am disappointed and frustrated to read a text in such need of a profound grammar and style revision.In my opinion, the current version of the MS makes a disservice to its scientific contents.2-In the Abstract, the authors mention that "Tet interacts directly with BAP55 and Stat92E, facilitating recruitment of the PBAP complex to the dpp promoter and activating Dpp expression".However, they do not demonstrate any binding of the complex to the dpp gene.I believe the work would be much improved if the authors could show the binding of the complex to the dpp promoter or to an enhancer of the gene.I would suggest to do chromatin immunoprecipitation with S2 cells or similar, provided they express dpp and they respond to tet-stat92E-bap55 interactions (they use this cell type to search for tet-associated proteins, Fig. 3A).Alternatively, they could try an EMSA (electrophoretic mobility shift assay) with suitable dpp sequences.Minor points: 1-The Title of the MS reads "A novel dioxygenase-independent tet function...".I am not sure if this is really a novel function.I have found several published articles in which some of the vertebrate Tet genes are required to complex transcription factors to their target genes.2-Please explain the meaning of IGS.Do you refer to escort cells?3-"In the germarium, two or three GSCs directly contact cap cells and anterior IGS cells, which form the niche for controlling their self-renewal, whereas CBs, mitotic cysts and early 16-cell cysts are wrapped up by the long cellular processes of posterior IGS cells, which constitute the niche for promoting these GSC progeny differentiation".Consider improving the grammar.Also, which renewal are you refereeing to?I assume it is GSC renewal, but it is not clear from the sentence.4-"Furthermore, Jak-Stat signaling controls Dpp expression in cap cells to maintain GSC self renewal".Please cite, here and in the rest of the text, Lopez-Onieva et al.Development 2008 (Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary).5-"In this study, two independent transgenic shRNA lines were used to knock down Tet in adult cap cells (bab1ts>Tet-KD1 and bab1ts>Tet-KD2)".Careful with this statement (and also in other sections of the text).In addition to cap cells, bab1-Gal4 is also expressed in terminal filament cells and in escort cells.6-I would not claim that LamC is "specifically expressed in TF and cap cells".It is enriched in these cell types, while also presenting lower levels in other cells of the germarium.7-Arrowhead in Fig. 1K seems to be pointing to the ring-canal associated fusome material of a 4-cell cyst recently divided, not to a CB spectrosome.8-When referring to the BAC transgenic strain for Bap111-GFP, please specify.Consider stating that Bap111-GFP is an inframe insertion in the genomic locus of a BAC transgene.Therefore, the flies have the two endogenous bap111 copies plus the BAC's babp111-GFP.9-"To determine which complex is involved in controlling dpp expression, we sought to knock down the expression of brm, Bap170, Bap180, and osa in adult niche cells, brm-KD, Bap170-KD, Bap180KD, and osa-KD.brm-KD, Bap170-KD, Bap180KD, and osa-KD significantly decrease GSCs compared to the control".Please amend.10-Fig.5B: Why does the input Flag-tet-CF-1 have so little amount compared to the other lines?Please explain in the fig.legend, particularly so when the IPed Flag-tet-CF-1 line has plentiful.Why does the IPed Flag-tet-CF-4 line show two bands?11-Fig.5D, F: why are there so many bands of Myc-Tet-C (D, E) and HA-Stat92E (F)?What do the asterisks mean in D-F? 12-Bap55-Stat92E fusion: How was this fusion made?Details in the M+M are scarce and confusing, at least to me, as I do not know if it is just an in-frame fusion of both full length ORFs or something different.This is one of the most important experiments of the MS and should be reported with greater detail.13-Fig.5G: I assume the difference in the number of GSCs/germarium of wt and tet-KD-1 is statistically significant.The authors may want to show that in the graph, as they have done with 5K and 5L.14-Human tet3: It would be interesting to show if a dead version of the Tet3 protein is also able to rescue.

Referee #3:
This manuscript demonstrates a novel function of TET proteins, independent of catalytic activity, to act as a scaffold protein the associate the PBAP chromatin remodelling complex with STAT92E in order to activate expression of Dpp in the Drosophila ovarian stem cell niche.This is a highly significant study from 2 aspects: 1) association of a novel TET activity (and also showing conservation of function between Drosophila and human) but also 2) it has been long known that STAT and Dpp signalling somehow interact in the stem cell niche but the mechanism has been unclear.This study provides mechanistic evidence for this interaction.The manuscript is well written and data are clearly presented.Concerns that need to be addressed: 1) The authors state "The Tet-KD1 and Tet-KD2 germaria significantly decrease GSCs compared to the control although the control and Tet knockdown germaria have comparable CBs (Fig. 1C and 1D)."Why does a loss of GCSs also not flow on the result in a loss of CBs?
2) The Materials and Methods state that all of the statistical tests are T-tests.Given that the graphs all show more than two groups should the statistical tests not all have been ANOVAs?3) The authors state "Like Tet overexpression, niche-specific hTET3 overexpression (hTET3-OE) alone can slightly increase dpp mRNA expression in cap cells and pMad expression in GSCs but does not have obvious effect on GSCs (Fig. 6A-F)."However, Fig. 6F does not show an increase in the hTet3-OE column compared to WT control, and 6D does not show an increase in pMad expression compared to WT. 4) In the discussion could the authors please provide further statements regarding the interaction between the PBAP complex and STAT92E.Does STAT92E need to be prior phosphorylated via JAK activity before interacting with STAT92E.Additional minor concerns are: 1) In Figure 1B could a germ cell marker also be shown to conclusively show that TET-EGFP is not expressed in germ cells Referee #1, additional comment: One point for discussion is the reviewer comment about T-tests vs ANOVA.Although the figures do show quantification of more than two groups, the relevant statistical tests are the specific pairwise comparisons between wildtype vs each of the experimental genotypes.ANOVA would be more appropriate if the experimenters had no a priori knowledge of which pairwise comparison would be most relevant or interesting (e.g. a difference between two of the experimental genotypes would be just as important as a difference between a control and an experimental).Since this is not the case, I believe T-tests are most appropriate.However, since they are performing multiple T-tests in the same experiment, one could argue that a multiple comparison correction such as bonferroni should be applied, but I don't think that is commonly done in this case.

Referee #1:
In this study, the authors investigate the role of the Tet protein in Drosophila ovarian germline stem cell maintenance.They find that Tet is expressed in somatic cells and that RNAi knockdown specifically in the bab1+ population (which provide niche function for the GSCs) impairs GSC selfrenewal.Further investigation reveals that this is due to a non-enzymatic scaffolding function of the tet protein that brings stat92E together with the BAP/PBAP complex protein Bap55.Building on a previous study that found that Jak/Stat signaling activates dpp expression in the bab1+ population, they show that RNAi knockdown of tet or the PBAP proteins (but not osa, which is specific for the BAP complex) reduces dpp expression, BMP signaling in GSCs, and GSC number.Rescue experiments using a wildtype or enzymatic dead allele of tet and in vitro co-IP experiments provide substantial support for the idea that tet has a non-enzymatic, scaffolding role in this process.Taken together, their data advance our understanding of the molecular mechanisms that contribute to Drosophila germline stem cell self renewal and the evidence for a non-enzymatic function of tet will be of interest more broadly.However, several concerns should be addressed before publication: Response: Thank this reviewer for recognizing the significance and importance of our study and providing some constructive comments.
1.The data provided suggest that osa is required for GSC maintenance but not for dpp expression of BMP signaling within GSCs.What is the explanation for this?Also, since the conclusion from these data that the BAP complex is not required for dpp expression is based on this one RNAi line, the authors should confirm that the osa RNAi is, indeed, effectively knocking down osa transcript, as expected.
Response: There are two BRM-containing chromatin remodeling complexes, PBAP and BAP, in Drosophila.Although PBAP and BAP components are required to maintain GSCs, knocking down the PBAP/BAP-shared components and PBAP-specific component disrupts BMP signaling in GSCs but not E-cadherin accumulation in the stem cell-niche junction.However, knocking down Osa causes the downregulation of E-cadherin in the stem cell-niche junction, suggesting that Osa regulates Ecadherin expression in the niche independently of the BAP complexes.Using qPCR, we have confirmed that the osa RNAi strain effectively reduces osa mRNA levels when driven by tub-Gal4 (Fig. S1F).
2. The mass spectrometry approach associated with the data in Fig. 3A do not appear to be described in the methods.This should be added.

Response:
The information has been added to the revised manuscript as requested.

Throughout the figures, it is not clear what is meant by "relative intensity". What are these intensity measurements relative to?
The methods state that internal imaging controls were measured, but do not provide details about what these internal controls are.Also, the method of controlling for background signal (e.g. a background subtraction or correction) should be described.

Response:
The term "relative intensity" in this context is referred to as the intensity to the control.We have included a description of these procedures in the Methods section.
4. The method for unambiguously identifying CBs is not well described.Related to this, the object that the arrowhead in Fig. 1C is pointing to is not a very clear example of a rounded spectrosome that is clearly distinct from the other hts structures nearby.

28th Dec 2023 1st Authors' Response to Reviewers
Response: CBs are defined as single germ cells with a round fusome and at least one cell distance from cap cells.We have replaced Fig. 1C with high quality images.
5. In Fig. S3, the Bap111 channel should be shown separately.The signal is dim and hard to appreciate in the multicolor image.In addition, these data show that tet is not essential for maintaining bap111 expression but do not provide strong support for the statement that Tet is not essential for maintaining the expression level of the whole BAP and PBAP complexes, as stated at the bottom of page 8.

Response:
We have included single-channel images as suggested.We agree that Bap111-GFP does not fully represent overall expression levels of the entire PBAP complex.Thus, we have revised our statement accordingly.

Referee #2:
In this work, the authors report the function of the Tet protein in the Drosophila ovarian stem cell niche.They convincingly show that an enzymatic-dead Tet is necessary to ensure proper dpp transcription, an essential gene for germline stem cell (GSC) maintenance in the niche.Tet is required to bring together components of the PBAP complex and the Stat92E transcription factor.In their model, complexed Tet-PBAP-Stat bind to the dpp gene to activate its transcription and, thus, to regulate niche activity.I believe the biological question being investigated to be of interest.However, in my opinion the novelty of the findings appears somewhat limited as to warrant publication in a journal with a broad readership such as The EMBO J. Dioxygenase activity-independent Tet functions are well established in the field, as is the relationship between Jak/Stat and dpp transcription in the GSC niche.The MS does report some advancements, such as the demonstration of Tet-Stat92 and Tet-Bap55 physical interactions and the role of the complex in regulating dpp mRNA levels in the cap cells.Nevertheless, it fails to provide evidence for the binding of the complex to the dpp gene.

Response:
We appreciate the recognition of the significance of our work and the constructive comments from this reviewer.

Major points: 1-I have a serious issue with the current presentation of the MS. To follow is a list (by no means exhaustive) of some of the problems I detected: a) I have found typos and spelling mistakes in the text and figures (for instance, Fig 1L "reseuce" instead of "rescue"; Fig 2A enzyme, not ensyme). b)
In several instances, the authors talk of "decrease GSCs" or "comparable CBs".I assume they refer to reduced GSC numbers or comparable CB numbers.c) Sentences with very similar contents are repeated throughout the text (for instance, in the Abstract: "Tet acts as a scaffold protein, bringing together the PBAP chromatin remodeling complex and the Stat92E transcription factor to activate the expression of BMP-like Dpp in the niche" and, two sentences later, "Mechanistically, Tet interacts directly with BAP55 and Stat92E, facilitating recruitment of the PBAP complex to the dpp promoter and activating Dpp expression".Or in the first paragraph of the Discussion "Tet functions as a protein scaffold to recruit the PBAP complex to Stat92E by directly interacting with Bap55 and Stat92E.Bap55-Stat92E can bypass the requirement of Tet in the niche to maintain BMP signaling and GSC self-renewal" and later in the paragraph "we propose that Tet acts as a protein scaffold, functioning independently of its enzymatic activity.It facilitates the interaction between the PBAP complex and Stat92E, leading to the activation of dpp expression in the niche".Or later in the Discussion "In this study, we show that TET functions as a protein scaffold independently of its enzymatic activity to recruit the PBAP complex to Stat92E to activate Dpp expression in the niche for controlling GSC self-renewal in the Drosophila ovary.In this study, we have used a combination of genetics and biochemistry to demonstrate that Tet functions independently of its dioxygenase activity in the niche to control GSC self-renewal by regulating BMP signaling".i) Treatments should also be clearly reported.The M+M section has a general statement that does not provide sufficient detail "newly eclosed flies at room temperature were cultured at 29{degree sign}C for the specified days before phenotypic analysis".As you may hint from my words, I am disappointed and frustrated to read a text in such need of a profound grammar and style revision.In my opinion, the current version of the MS makes a disservice to its scientific contents.
Response: Thank the reviewer for taking time to identify errors and provide suggestions.We have made the following revisions as suggested: correcting the typos (1a, 1b), reversing the statements for clarity (1c), adding the reference (1d), resolving the genotype notation issue (1g).Regarding point 1e, the absence of Fig S1 in the reviewer's download might be due to an issue in the journal's website.Regarding point 1f, we acknowledge that our initial submission did not closely follow the EMBO format.In the new version, we have updated the figure legends accordingly.For point 1h, we used luc-KD as the control, and we have provided a description of this in the revised manuscript.Concerning point 1i, we have added a detailed description as suggested.
2-In the Abstract, the authors mention that "Tet interacts directly with BAP55 and Stat92E, facilitating recruitment of the PBAP complex to the dpp promoter and activating Dpp expression".However, they do not demonstrate any binding of the complex to the dpp gene.I believe the work would be much improved if the authors could show the binding of the complex to the dpp promoter or to an enhancer of the gene.I would suggest to do chromatin immunoprecipitation with S2 cells or similar, provided they express dpp and they respond to tet-stat92E-bap55 interactions (they use this cell type to search for tet-associated proteins, Fig. 3A).Alternatively, they could try an EMSA (electrophoretic mobility shift assay) with suitable dpp sequences.

Response:
We have conducted ChIP-qPCR experiments to show that Tet-C-GFP, Bap55-HA, and Stat92E-HA are directly associated with a previously identified dpp2.0 enhancer in the dpp genomic region, which is known to be required for controlling dpp expression in cap cells (Fig. 6).Interestingly, we have also observed a decrease in the enrichment of Bap55-HA and Stat92E-HA pulldown when Tet was knocked down.Our new experimental results show that Tet, Bap55 and Stat92E are indeed co-localized to the dpp enhance to control its expression in the niche.
Minor points: 1-The Title of the MS reads "A novel dioxygenase-independent tet function...".I am not sure if this is really a novel function.I have found several published articles in which some of the vertebrate Tet genes are required to complex transcription factors to their target genes.
Response: In our study, we discovered that Tet acts as a scaffolding protein, connecting the important PBAP complex with Jak/Stat signaling to control dpp expression in the Drosophila ovarian stem cell niche.This finding is novel and distinct from what has been previously reported in the literature.

2-Please explain the meaning of IGS. Do you refer to escort cells?
Response: Inner germarial sheath (IGS) cells are also called escort cells.We updated this in the revised manuscript.

3-"In the germarium, two or three GSCs directly contact cap cells and anterior IGS cells, which form
the niche for controlling their self-renewal, whereas CBs, mitotic cysts and early 16-cell cysts are wrapped up by the long cellular processes of posterior IGS cells, which constitute the niche for promoting these GSC progeny differentiation".Consider improving the grammar.Also, which renewal are you refereeing to?I assume it is GSC renewal, but it is not clear from the sentence.

Response:
The sentence is revised.

4-"Furthermore, Jak-Stat signaling controls Dpp expression in cap cells to maintain GSC self renewal". Please cite, here and in the rest of the text, Lopez-Onieva et al. Development 2008 (Jak/Stat signalling in niche support cells regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary).
Response: The inadvertently missed reference is cited as it should.

6-I would not claim that LamC is "specifically expressed in TF and cap cells". It is enriched in these cell types, while also presenting lower levels in other cells of the germarium.
Response: Corrected.

7-Arrowhead in Fig. 1K seems to be pointing to the ring-canal associated fusome material of a 4-cell cyst recently divided, not to a CB spectrosome.
Response: The image has been replaced.

8-When referring to the BAC transgenic strain for Bap111-GFP, please specify. Consider stating that
Bap111-GFP is an in-frame insertion in the genomic locus of a BAC transgene.Therefore, the flies have the two endogenous bap111 copies plus the BAC's babp111-GFP.
Response: It has been modified accordingly.9-"To determine which complex is involved in controlling dpp expression, we sought to knock down the expression of brm, Bap170, Bap180, and osa in adult niche cells, brm-KD, Bap170-KD, Bap180KD, and osa-KD.brm-KD, Bap170-KD, Bap180KD, and osa-KD significantly decrease GSCs compared to the control".Please amend.Response: This may be due to the instability of the Flag-Tet-CF-1 protein.However, after enrichment with Flag antibody, we were able to detect a higher amount of Flag-Tet-CF-1, indicating the successfulness of the Co-IP experiment.A previous study reported that Tet proteins can undergo cleavage (Wang, Y. and Y. Zhang, 2014.Cell Reports 6(2): 278-284.PMID: 24412366).The presence of two bands in Flag-tet-CF-4 may be attributed to the generation of a smaller band through protein cleavage, which is enriched by the Flag antibody.Therefore, it is not evident in the Input, but it can be enriched after IP.

11-Fig. 5D, F: why are there so many bands of Myc-Tet-C (D, E) and HA-Stat92E (F)? What do the asterisks mean in D-F?
Response: Asterisks (*s) indicate these bands formed due to protein instability and degradation.We have added a description regarding the asterisks in the figure legend.

12-Bap55-Stat92E fusion: How was this fusion made? Details in the M+M are scarce and confusing, at least to me, as I do not know if it is just an in-frame fusion of both full length ORFs or something different. This is one of the most important experiments of the MS and should be reported with greater detail.
Response: We have added a detailed protocol in the Methods section regarding the generation of the Bap55-Stat92E fusion.
13-Fig.5G: I assume the difference in the number of GSCs/germarium of wt and tet-KD-1 is statistically significant.The authors may want to show that in the graph, as they have done with 5K and 5L.
Response: It is statistically significant, which has been updated in the figure.

14-Human tet3: It would be interesting to show if a dead version of the Tet3 protein is also able to rescue.
Response: Yes, it would be nice to have the experiment done.Our experiments one the fly Tet has implied that this would be the case.The allowed revision time is not sufficient for us to construct the new transgenic fly strain and conduct genetic crosses.

Referee #3:
This manuscript demonstrates a novel function of TET proteins, independent of catalytic activity, to act as a scaffold protein the associate the PBAP chromatin remodelling complex with STAT92E in order to activate expression of Dpp in the Drosophila ovarian stem cell niche.This is a highly significant study from 2 aspects: 1) association of a novel TET activity (and also showing conservation of function between Drosophila and human) but also 2) it has been long known that STAT and Dpp signalling somehow interact in the stem cell niche but the mechanism has been unclear.This study provides mechanistic evidence for this interaction.The manuscript is well written and data are clearly presented.Concerns that need to be addressed: Response: We appreciate the valuable time and the positive comments.

1) The authors state "The Tet-KD1 and Tet-KD2 germaria significantly decrease GSCs compared to the control although the control and Tet knockdown germaria have comparable CBs (Fig. 1C and 1D)." Why does a loss of GCSs also not flow on the result in a loss of CBs?
Response: This reviewer raised an interesting question.This phenomenon has been observed many times by our lab.We speculate that as the GSC number decreases, the CB might also slow down its differentiation process, including cyst formation.

2) The Materials and Methods state that all of the statistical tests are T-tests. Given that the graphs all show more than two groups should the statistical tests not all have been ANOVAs?
Response: Although there are multiple groups in some of the graphs, we have only done the pairwise comparison, so we only use Student's t-test for our statistical analysis.For example, a RNAi group is compared with the luc-KD control group to determine the significance of the change induced by gene knockdown.In the revised manuscript, we have indicated with arrows in the graphs the two groups used for t-tests.

3) The authors state "Like Tet overexpression, niche-specific hTET3 overexpression (hTET3-OE) alone can slightly increase dpp mRNA expression in cap cells and pMad expression in GSCs but does
not have obvious effect on GSCs (Fig. 6A-F)."However, Fig. 6F does not show an increase in the hTet3-OE column compared to WT control, and 6D does not show an increase in pMad expression compared to WT.

4) In the discussion could the authors please provide further statements regarding the interaction between the PBAP complex and STAT92E. Does STAT92E need to be prior phosphorylated via JAK activity before interacting with STAT92E.
Response: A statement is provided as suggested.

Additional minor concerns are: 1) In Figure 1B could a germ cell marker also be shown to conclusively show that TET-EGFP is not expressed in germ cells
Response: We use co-staining of Tet-EGFP and Vasa to show that Tet-EGFP is absent in Vasapositive germ cells.

Referee #1, additional comment:
One point for discussion is the reviewer comment about T-tests vs ANOVA.Although the figures do show quantification of more than two groups, the relevant statistical tests are the specific pairwise comparisons between wildtype vs each of the experimental genotypes.ANOVA would be more appropriate if the experimenters had no a priori knowledge of which pairwise comparison would be most relevant or interesting (e.g. a difference between two of the experimental genotypes would be just as important as a difference between a control and an experimental).Since this is not the case, I believe T-tests are most appropriate.However, since they are performing multiple T-tests in the same experiment, one could argue that a multiple comparison correction such as bonferroni should be applied, but I don't think that is commonly done in this case.
Response: Thank you very much for this comment!We have addressed this issue earlier accordingly.

6th Feb 2024 1st Revision -Editorial Decision
Dear Dr Ting Xie, Thank you for submitting your revised manuscript (EMBOJ-2023-115586R) to The EMBO Journal.Your amended study was sent back to the three referees for their scientific re-evaluation, and we have received detailed comments from all of them, which I enclose below.
As you will see, the experts state that the work has been substantially improved by the revisions and they are now in favour of publication, pending minor revision.
Thus, we are pleased to inform you that your manuscript has been accepted in principle for publication in The EMBO Journal.
Please consider the remaining minor comment of referee #2 regarding sample annotation carefully and amend the manuscript text accordingly.Also, we now need you to take care of a number of issues related to formatting and data presentation as detailed below, which should be addressed at re-submission.
Please contact me at any time if you have additional questions related to below points.
Thank you for giving us the chance to consider your manuscript for The EMBO Journal.I look forward to your final revision.
Again, please contact me at any time if you need any help or have further questions.

Best regards, Daniel Klimmeck
Daniel Klimmeck PhD Senior Editor The EMBO Journal ******* Formatting changes required for the revised version of the manuscript: >> Author Contributions: Please remove the author contributions information from the manuscript text.Note that CRediT has replaced the traditional author contributions section as of now because it offers a systematic machine-readable author contributions format that allows for more effective research assessment.and use the free text boxes beneath each contributing author's name to add specific details on the author's contribution.
More information is available in our guide to authors.https://www.embopress.org/page/journal/14602075/authorguide>>Manuscript composition: the order of the manuscript sections should be: .. >> Consider additional changes and comments from our production team as indicated below: -Figure Legends (main + EV): Please note that in figure 6f; there is a mismatch between the annotated p values in the figure legend and the annotated p values in the figure file that should be corrected.

Referee #2:
The AUs have addressed most of my concerns and my major criticism (the binding of the Stat-Tet-Bap55 complex to the dpp gene) satisfactorily.
In the new quantification by qPCR of the efficiency of the RNAi approach, the AUs ought to explain which tissue they isolated the mRNA from.I assume it was from ovary tips, as the reduction in mRNA levels was quite significant for all the genes, and using the bab1-Gal4 line to drive the shRNA would only affect early oogenesis.

Referee #3:
I am now satisfied that the authors have made substantial improvements to the manuscript and addressed all of my concerns.

19th Feb 2024 2nd Authors' Response to Reviewers
The authors addressed the remaining editorial issues.

23rd Feb 2024 2nd Revision -Editorial Decision
Dear Dr Ting Xie, Thank you for submitting the revised version of your manuscript.I have now evaluated your amended manuscript and concluded that the remaining minor concerns have been sufficiently addressed.
I am pleased to inform you that your manuscript has been accepted for publication in the EMBO Journal.Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
You may qualify for financial assistance for your publication charges -either via a Springer Nature fully open access agreement or an EMBO initiative.Check your eligibility: https://www.embopress.org/page/journal/14602075/authorguide#chargesguideShould you be planning a Press Release on your article, please get in contact with embo_production@springernature.com as early as possible in order to coordinate publication and release dates.
On a different note, I would like to alert you that EMBO Press offers a format for a video-synopsis of work published with us, which essentially is a short, author-generated film explaining the core findings in hand drawings, and, as we believe, can be very useful to increase visibility of the work.This has proven to offer a nice opportunity for exposure i.p. for the first author(s) of the study.Please see the following link for representative examples and their integration into the article web page: https://www.embopress.org/video_synopseshttps://www.embopress.org/doi/full/10.15252/embj.2019103932 Please let me know, should you be interested to engage in commissioning a similar video synopsis for your work.According operation instructions are available and intuitive.
If you have any questions, please do not hesitate to contact the Editorial Office.------------------------------------------------>>> Please note that it is The EMBO Journal policy for the transcript of the editorial process (containing referee reports and your response letter) to be published as an online supplement to each paper.If you do NOT want this, you will need to inform the Editorial Office via email immediately.More information is available here: https://www.embopress.org/transparentprocess#Review_Process

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d) There is one duplicated reference (Delatte et al 2016).e) Fig. S1 is missing from this version.f) The writing of the Fig. legends lacks detail and is redundant (every single panel containing statistical analyses has the following (or very similar) statement "Student's t test: ***p< 0.001; n. s., no significance".Or the repetition of the meaning of a dash line, arrows or arrowheads in all the panels of the same figure.This makes fig.legends very repetitive) g) Gene nomenclature is not consistent throughout (for instance, there is Bab1-Gal4, bab-Gal4, bab1-Gal4; or there is tet-RR-wt and tet-wt, tet-Dead and tet-ED) h) The full genotypes of the different experiments are not reported (what is "wt" in the different experiments?Canton S or Oregon flies?or Gal4 lines without UAS transgenes, or viceversa?).
: Corrected.10-Fig.5B: Why does the input Flag-tet-CF-1 have so little amount compared to the other lines?Please explain in the fig.legend, particularly so when the IPed Flag-tet-CF-1 line has plentiful.Why does the IPed Flag-tet-CF-4 line show two bands?
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